Method of culturing microorganism

ABSTRACT

In cultivating a microorganism having nitrile hydratase production ability, by allowing at least one of a ketose, such as fructose, and a sugar alcohol, such as mannitol, to be present, growth inhibition by cobalt ion is prevented, thereby achieving cultivation at a high concentration and with a high activity.

TECHINICAL FIELD

The present invention relates to a method of producing culture microbial cells having high nitrile hydratase enzyme activity within a short period and at a high yield.

BACKGROUND ART

In recent years, from the viewpoint of energy conservation, renewed activity has been seen in attempts to utilize microorganisms or their enzymes as a biocatalyst for matter production. Nitrile hydratase is known as an enzyme which hydrates nitrites to thereby produce the corresponding amides. In particular, it is useful as a catalyst to produce acrylamide from acrylonitrile.

As microorganisms having nitrile hydratase production ability, for example, there are known bacteria belonging to genus Nocardia [see Japanese Patent Application Laying-Open (kokai) No. 54-129190], genus Rhodococcus [see Japanese Patent Application Laying-Open (kokai) No. 2-470], genus Rhizobium [see Japanese Patent Application Laying-Open (kokai) No. 5-236977], genus Klebsiera [Japanese Patent Application Laying-Open (kokai) No. 5-30982], genus Aeromonas [Japanese Patent Application Laying-Open (kokai) No. 5-30983], genus Agrobacterium [Japanese Patent Application Laying-Open (kokai) No. 8-154691], genus Bacillus [Japanese Patent Application Laying-Open (kokai) No. 8-187092], genus Pseudonocardia [Japanese Patent Application Laying-Open (kokai) No. 8-56684], and the like. In each of the above-mentioned publications, a method of cultivating microorganisms which produce nitrile hydratase activity is described.

Further, with respect to use of carbon source in cultivating, it is described in the section “Effect of carbon sources” of Applied Microbiology and Biotechnology (1991) 34:783-788 that “in producing nitrile hydratase by use of R. rhodochroits J1, the most suitable carbon source is 2% glucose.”

DISCLOSURE OF THE INVENTION

None of the methods described in the above-mentioned publications are industrially practical as a method of obtaining culture microbial cells having nitrile hydratase activity within a short period and at a high yield.

For the purpose of enzyme induction, cobalt ions are added to culture solution according to the method described in Japanese Patent Application Laying-Open (kokai) No. 2-470, though it is difficult by employing such cobalt ion concentration to always effect stable obtainment of culture microbial cells having nitrile hydratase enzyme activity within a short period and at a high yield. Further, addition of a large amount of cobalt ions to the medium will cause remarkable growth inhibition, and, on the contrary, it is not possible to obtain culture microbial cells having nitrile hydratase activity within a short period and at a high yield.

In addition, in order to obtain culture microbial cells having nitrile hydratase activity within a short period and at a high yield, glucose or sucrose is considered preferable as a carbon source, and use of ketoses such as fructose or sugar alcohols such as mannitol has conventionally been considered scientifically impossible.

The object of the present invention is to provide a method of obtaining culture microbial cells having nitrile hydratase activity within a short period and at a high yield which solves the above problems.

The present inventors have conducted intensive studies on culture conditions to industrially obtain microbial cells having nitrile hydratase activity within a short period and at a high yield. As a result, the inventors found it possible to obtain highly active microbial cells as well as to reduce growth inhibition even in the presence of a large amount of cobalt ions in culture solution by allowing the presence of a ketose, e.g. fructose, and/or a sugar alcohol, e.g. mannitol, in the culture solution. Thus, the present invention has been accomplished.

Namely, the present invention is a method of cultivating a microorganism having nitrile hydratase production ability in a medium substrate containing a sugar alcohol and/or a ketose, and a cobalt ion. Herein, the cobalt ion concentration is preferably 15 mg/L or more in terms of CoCl₂. Additionally, the sugar alcohol is preferably represented by the following general formula (I):

wherein n represents an integer from 2 to 4. Furthermore, the ketose is represented by the following general formula (II):

wherein n represents 3.

According to the present invention, a sugar alcohol and/or a ketose are used as a carbon source. However, it is not conventionally known that the presence of these sugar compounds in a culture solution allows the reduction of growth inhibition attributable to cobalt ion.

The present invention will hereinafter be described in detail.

The microorganisms of interest according to the present invention are not particularly limited, as long as they have nitrile hydratase production ability. Specifically, preferable examples of the microorganisms include Nocardia sp. N-775 described in Japanese Patent Examined Publication (kokoku) No. 56-17918, Rhodococcus rhodochrous J-1 described in Japanese Patent Examined Publication (kokoku) No. 06-55148, Klebsiella sp. MCI2609 described in Japanese Patent Application Laying-Open (kokai) No. 05-30982, Aeromonas sp. MCI2614 described in Japanese Patent Application Laying-Open (kokai) No. 05-30983, Citrobacter freundii MCI2615 described in Japanese Patent Application Laying-Open (kokai) No. 05-30984, Agrobacterium rhizogenes IAM13570 and Agrobacterium tumefaciens described in Japanese Patent Application Laying-Open (kokai) No. 05-103681, Xanthobacter flavas JCM1204 described in Japanese Patent Application Laying-Open (kokai) No. 05-161495, Erwinia nigrifluens MAFF03-01435, Enterobacter sp. MCI2707 described in Japanese Patent Application Laying-Open (kokai) No. 05-236975, Streptomyces sp. MCI2691 described in Japanese Patent Application Laying-Open (kokai) No. 05-236976, Rhizobium sp. MCI2610, Rhizobium sp. MCI2643, Rhizobium loti IAM13588, Rhizobium legminosarum IAM12609 and Rhizobium merioti IAM12611 described in Japanese Patent Application Laying-Open (kokai) No. 05-236977, Candida guilliermondii NH-2, Pantoea agglomerans NH-3 and Klebsiella pneumoniae subsp. pneumoniae NH-26T2 described in Japanese Patent Application Laying-Open (kokai) No. 05-15384, Agrobacterium radiobacter SC-C15-1 described in Japanese Patent Application Laying-Open (kokai) No. 06-14786, Bacillus smithii SC-J05-1 described in Japanese Patent Application Laying-Open (kokai) No. 07-25494, Pseudonocardia thermophila ATCC19285 described in Japanese Patent Application Laying-Open (kokai) No. 08-56684, and Pseudonocardia thermophila JCM3095 described in Japanese Patent Application Laying-Open (kokai) No. 09-275978.

Additionally, the microorganisms of interest according to the present invention also include transformants which may be any host wherein a nitrile hydratase gene cloned from the above microorganisms is expressed.

Preferable examples thereof include MT-10822 bacteria strain (FERM BP-5785) described in U.S. Pat. No. 5,807,730 wherein Escherichia coli is used as a typical host.

As a medium substrate, examples of a carbon source include a sugar alcohol represented by the general formula (I) and/or a ketose represented by the general formula (II). Specifically, fructose, mannitol and sorbitol are preferable. These carbon sources can also be used in a suitable combination thereof.

(wherein n represents an integer from 2 to 4)

-   -   (wherein n represents 3)

Any supply source of cobalt ion may be employed as long as it is a divalent or trivalent cobalt salt. Examples thereof include cobalt chloride, cobalt sulfate, cobalt acetate, cobalt bromide, and cobalt borate. In addition, vitamin B₁₂ or the like may be the cobalt source. Further, the cobalt ion concentration is 15 mg/L or more in terms of CoCl₂, preferably 15 to 300 mg/L.

Nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate; organic nutritive source such as yeast extract, meat extract, malt extract, peptones, and Soybean hydrolyzate; and inorganic salts such as phosphates, magnesium salts, potassium salts, sodium salts, iron salts, cobalt salts, and other trace mineral salts, are selected for the preparation of the medium. As needed, an enzyme inducer such as urea or its derivatives may be added.

Further, if necessary, the carbon source, cobalt ions or the like may be added in portions while cultivating.

While cultivating, the pH is to be kept normally at 5 to 10, preferably 7 to 9. The cultivation temperature is normally 25 to 50° C., preferably 30 to 40° C., and the cultivation is aerobically conducted for 1 to 3 days.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically by referring to the following Examples. However, the present invention is not limited to these Examples.

EXAMPLE 1

(1) Cultivating Microbes

{circle around (1)} Pre-culture Conditions:

(Medium Composition)

-   fructose 2% (w/v), polypeptone 5% (w/v) (Nihon Pharmaceutical Co.,     Ltd.), yeast extract 0.3% (w/v) (Oriental Yeast Co., Ltd.), KH₂PO₄     0.1% (w/v), K₂HPO₄ 0.1% (w/v), MgSO₄ 7H₂O 0.1% (w/v), pH 7     (Culture Method)

100 ml of the medium was dispensed into a 500 ml Erlenmeyer flask and the flask was sealed with a cotton plug. The medium was sterilized by an autoclave for 20 minutes at 121° C. Then, Rhodococcus rhodochrous J-1 strain (FERM BP-1478) was inoculated thereto and the mixture was subjected to shake cultivation for 48 hours at 30° C.

{circle around (2)} Main Culture Conditions

(Medium Composition)

Initial medium: yeast extract 0.2% (w/v), KH₂PO₄ 0.1% (w/v), K₂HPO₄ 0.1% (w/v), MgSO₄.7H₂O 0.1% (w/v), CoCl₂.6H₂O 0.002% (w/v), ammonium sulfate 0.025% (w/v), fructose 2% (w/v), urea 2% (w/v), ethanol 0.4% (v/v), Pluronic L61 0.1% (w/v) (Asahi Denka Kogyo K.K.), pH 7

After-addition medium: fructose 20% (w/v), ethanol 5% (v/v), ammonium sulfate 6% (w/v), pH 6.5

(Culture Method)

Two liters of the initial medium was dispensed into a 3-liter mini jar fermenter (Takasugi Seisakusho) and sterilized by an autoclave for 20 minutes at 121° C. However, apart from the above, fructose, ethanol and urea were aseptically filtrated (using 0.45-micron filter paper manufactured by Advantec Toyo Kaisha, Ltd.) and then added to the medium. After termination of the above pre-culture, 20 ml of the pre-cultivated solution was inoculated thereto and the resultant medium was cultivated for 44 hours at 30° C. under the conditions of an in-tank pressure of 0.098 MPa, a stirring rate of 600 rpm, an airflow quantity of 1 vvm, and pH 7.

In addition, from 20 hours onward after the start of the cultivation, the after-addition medium was added at a flow rate of 20 ml/hr until the end of the cultivation.

(2) Determination of Nitrile Hydratase Activity

0.1 ml of the culture solution and 4.90 ml of 1/20M phosphate buffer solution (pH 7.7) were mixed, and a further 5 ml of 1/20M phosphate buffer solution (pH 7.7) containing 5.0% (w/v) of acrylonitrile was added thereto, and the mixture was then reacted for 10 minute at 10° C. After termination of the reaction, microbial cells were filtered and separated by centrifugation. Acrylamide in the supernatant was quantified by gas chromatography (GC-14B manufactured by Shimadzu Corporation). Analysis was carried out under the following conditions: use of 1 m glass column packed with Porapack PS (column packing material manufactured by Waters Corporation); column temperature of 230° C.; use of FID at 250° C.

Hereinafter, one unit of enzyme activity in the above activity determination is defined as the conversion of 1 μmol of acrylonitrile into acrylamide in 1 minute. With respect to the activity, investigation was carried out per culture solution and per microbial cell. The results are shown in Table 1. In the Table, by taking 100 as the value for activity under the cultivation in the presence of 0.002% (w/v) of CoCl₂.6H₂O, the activity values under the other culture conditions are indicated as relative activity values (%).

EXAMPLES 2 to 5

Examples 2 to 5 were conducted in the same manner as Example 1, except for using main culture medium to which the following concentrations of CoCl₂.6H₂O were added instead of 0.002% (w/v) of CoCl₂.6H₂O: 0.005% (w/v) for Example 2, 0.01% (w/v) for Example 3, 0.02% (w/v) for Example 4, and 0.03% (w/v) for Example 5. The results are shown in Table 1.

EXAMPLES 6 and 7

Examples 6 and 7 were conducted in the same manner as Example 3, except for using both pre-culture and main culture mediums to which mannitol (Example 6) or sorbitol (Example 7) was added instead of fructose. The results are shown in Table 1.

COMPARATIVE EXAMPLE 1

Comparative Example 1 was conducted in the same manner as Example 1, except for using pre-culture and main culture mediums to which glucose was added instead of fructose. The results are shown in Table 1.

COMPARATIVE EXAMPLE 2 to 5

Comparative Examples 2 to 5 were conducted in the same manner as Comparative Example 1, except for using main culture medium to which the following concentrations of CoCl₂.6H₂O were added instead of 0.002% (w/v) of CoCl₂.6H₂O: 0.005% (w/v) for Comparative Example 2, 0.01% (w/v) for Comparative Example 3, 0.02% (w/v) for Comparative Example 4, and 0.03% (w/v) for Comparative Example 5. The results are shown in Table 1.

EXAMPLE 8

Example 8 was conducted in the same manner as Example 1, except for using Nocardia sp. N-775 strain (deposited as N-755 under the accession No. FERM BP-961) instead of Rhodococcus rhodochrous J-1 strain. The results are shown in Table 2.

EXAMPLE 9

Example 9 was conducted in the same manner as Example 8, except for using main culture medium to which 0.01% (w/v) of CoCl₂.6H₂O was added instead of 0.002% (w/v) of CoCl₂.6H₂O. The results are shown in Table 2.

EXAMPLES 10 and 11

Examples 10 and 11 were conducted in the same manner as Example 9, except for using pre-culture and main culture mediums to which mannitol (Example 10) or sorbitol (Example 11) was added instead of fructose. The results are shown in Table 2.

COMPARATIVE EXAMPLE 6

Comparative Example 6 was conducted in the same manner as Example 8, except for using pre-culture and main culture mediums to which glucose was added instead of fructose. The results are shown in Table 2.

COMPARATIVE EXAMPLE 7

Comparative Example 7 was conducted in the same manner as Comparative Example 6, except for using main culture medium to which 0.01% (w/v) of CoCl₂.6H₂O was added instead of 0.002% (w/v) of CoCl₂.6H₂O. The results are shown in Table 2.

TABLE 1 Activity per CoCl₂ Activity per culture Sugar Concentration microbial cell solution Example 1 Fru 0.002 100 100 Example 2 Fru 0.005 215 195 Example 3 Fru 0.01 252 229 Example 4 Fru 0.02 259 230 Example 5 Fru 0.03 259 231 Example 6 Mannitol 0.01 250 252 Example 7 Sorbitol 0.01 259 247 Comparative Example 1 Glc 0.002 100 18 Comparative Example 2 Glc 0.005 100 16 Comparative Example 3 Glc 0.01 48 6 Comparative Example 4 Glc 0.02 37 3 Comparative Example 5 Glc 0.03 7 0.2

TABLE 2 Activity per CoCl₂ Activity per culture Sugar Concentration microbial cell solution Example 8 Fru 0.002 100 100 Example 9 Fru 0.01 207 188 Example 10 Mannitol 0.01 217 197 Example 11 Sorbitol 0.01 230 209 Comparative Glc 0.002 83 15 Example 6 Comparative Glc 0.01 55 10 Example 7

EXAMPLE 12

(1) Cultivating Microbes

{circle around (1)} Pre-culture Conditions

(Medium Composition)

polypeptone 1% (w/v), yeast extract 0.5% (w/v), NaCl 0.5% (w/v), ampicillin.Na 100 μg/ml, pH7 (LB medium)

(Culture Method)

100 ml of the medium was dispensed into a 500-ml Erlenmeyer flask and the flask was sealed with a cotton plug. The medium was sterilized by an autoclave for 20 minutes at 121° C. Then, MT-10822 strain (FERM BP-5785) was inoculated thereto and the mixture was subjected to shake cultivation for 6 hours at 37° C.

{circle around (2)} Main Culture Conditions

(Medium Composition)

yeast extract 0.5% (w/v), polypepetone 2% (w/v), NaCl 0.06% (w/v), KCl 0.02% (w/v), MgCl₂.6H₂O 0.2% (w/v), CoCl₂.6H₂O 0.002% (w/v), MgSO₄.7H₂O 0.243% (w/v), fructose 4% (w/v), Pluronic L61 0.02% (w/v), pH 7

(Culture Method)

Two liters of the initial medium was dispensed into a 3-liter mini jar fermenter and sterilized by an autoclave for 20 minutes at 121° C. However, apart from the above, fructose and MgSO₄.7H₂O were aseptically filtrated (using 0.45-micron filter paper manufactured by Advantec Toyo Kaisha, Ltd.) and then added to the medium. After termination of the above pre-culture, 20 ml of the cultivated solution was inoculated thereto and the resultant medium was cultivated for 12 hours at 37° C. under the conditions of an in-tank pressure of 0.025 MPa, a stirring rate of 800 rpm, an airflow quantity of 1 vvm, and pH 7.

(2) Determination of Nitrile Hydratase Activity

Determination of nitrile hydratase activity was carried out in the same manner as in Example 1 (2). With respect to the activity, investigation was carried out per culture solution and per microbial cell. The results are shown in Table 3. In the Table, by taking 100 as the value for activity under the cultivation in the presence of 0.002% (w/v) of CoCl₂.6H₂O, the activity values under the other culture conditions are indicated as relative activity values (%).

EXAMPLE 13

Example 13 was conducted in the same manner as Example 12, except for using main culture medium to which 0.01% (w/v) of CoCl₂.6H₂O was added instead of 0.002% (w/v) of CoCl₂.6H₂O. The results are shown in Table 3.

EXAMPLES 14 and 15

Examples 14 and 15 were conducted in the same manner as Example 13, except for using main culture medium to which mannitol (Example 14) or sorbitol (Example 15) was added instead of fructose. The results are shown in Table 3.

COMPARATIVE EXAMPLE 8

Comparative Example 8 was conducted in the same manner as Example 12, except for using main culture medium to which glucose was added instead of fructose. The results are shown in Table 3.

COMPARATIVE EXAMPLE 9

Comparative Example 9 was conducted in the same manner as Comparative Example 8, except for using main culture medium to which 0.01% (w/v) of CoCl₂.6H₂O was added instead of 0.002% (w/v) of CoCl₂.6H₂O. The results are shown in Table 3.

TABLE 3 activity per CoCl₂ activity per culture Sugar Concentration microbial cell solution Example 12 Fru 0.002 100 100 Example 13 Fru 0.01 342 363 Example 14 Mannitol 0.01 528 113 Example 15 Sorbitol 0.01 436 363 Comparative Glc 0.002 87 87 Example 8 Comparative Glc 0.01 38 35 Example 9

INDUSTRIAL APPLICABILITY

Ketose such as fructose, sugar alcohols such as mannitol can reduce growth inhibition caused by cobalt ions in cultivating a microorganism having nitrile hydratase activity. As a result, it is possible to obtain microbial cells having nitrile hydratase enzyme activity within a short period and at a high yield 

1. A method of cultivating a microorganism having nitrile hydratase production ability in a medium substrate containing a sugar alcohol and/or ketose, and a cobalt ion.
 2. The method according to claim 1, wherein the sugar alcohol is represented by the general formula (I):

wherein n represents an integer from 2 to
 4. 3. The method according to claim 1, wherein the ketose is represented by the general formula (II):

wherein n represents
 3. 4. The method according to claim 1, wherein the concentration of cobalt is 15 mg/L or more in terms of CoCl₂.
 5. The method according to claim 1, wherein the concentration of cobalt ion is 20 mg/L or more in terms of CoCl₃. 